The multiple strategies of an insect herbivore to overcome plant cyanogenic glucoside defence

Cyanogenic glucosides (CNglcs) are widespread plant defence compounds that release toxic hydrogen cyanide by plant β-glucosidase activity after tissue damage. Specialised insect herbivores have evolved counter strategies and some sequester CNglcs, but the underlying mechanisms to keep CNglcs intact during feeding and digestion are unknown. We show that CNglc-sequestering Zygaena filipendulae larvae combine behavioural, morphological, physiological and biochemical strategies at different time points during feeding and digestion to avoid toxic hydrolysis of the CNglcs present in their Lotus food plant, i.e. cyanogenesis. We found that a high feeding rate limits the time for plant β-glucosidases to hydrolyse CNglcs. Larvae performed leaf-snipping, a minimal disruptive feeding mode that prevents mixing of plant β-glucosidases and CNglcs. Saliva extracts did not inhibit plant cyanogenesis. However, a highly alkaline midgut lumen inhibited the activity of ingested plant β-glucosidases significantly. Moreover, insect β-glucosidases from the saliva and gut tissue did not hydrolyse the CNglcs present in Lotus. The strategies disclosed may also be used by other insect species to overcome CNglc-based plant defence and to sequester these compounds intact

Autofluorescence-Based Identification and Functional Validation of Antennal Gustatory Sensilla in a Specialist Leaf Beetle

Herbivorous insects mainly rely on their sense of taste to decode the chemical composition of potential hosts in close range. Beetles for example contact and scan leaves with their tarsi, mouthparts and antennal tips, i.e., appendages equipped with gustatory sensilla, among other sensillum types. Gustatory neurons residing in such uniporous sensilla detect mainly non-volatile compounds that contribute to the behavioral distinction between edible and toxic plants. However, the identification of gustatory sensilla is challenging, because an appendage often possesses many sensilla of distinct morphological and physiological types. Using the specialized poplar leaf beetle (Chrysomela populi, Chrysomelidae), here we show that cuticular autofluorescence scanning combined with electron microscopy facilitates the identification of antennal gustatory sensilla and their differentiation into two subtypes. The gustatory function of sensilla chaetica was confirmed by single sensillum tip-recordings using sucrose, salicin and salt. Sucrose and salicin were found at higher concentrations in methanolic leaf extracts of poplar (Populus nigra) as host plant compared to willow (Salix viminalis) as control, and were found to stimulate feeding in feeding choice assays. These compounds may thus contribute to the observed preference for poplar over willow leaves. Moreover, these gustatory cues benefited the beetle’s performance since weight gain was significantly higher when C. populi were reared on leaves of poplar compared to willow. Overall, our approach facilitates the identification of insect gustatory sensilla by taking advantage of their distinct fluorescent properties. This study also shows that a specialist beetle selects the plant species that provides optimal development, which is partly by sensing some of its characteristic non-volatile metabolites via antennal gustatory sensilla

Lepidopteran defence droplets - a composite physical and chemical weapon against potential predators

Insects often release noxious substances for their defence. Larvae of Zygaena filipendulae (Lepidoptera) secrete viscous and cyanogenic glucoside-containing droplets, whose effectiveness was associated with their physical and chemical properties. The droplets glued mandibles and legs of potential predators together and immobilised them. Droplets were characterised by a matrix of an aqueous solution of glycine-rich peptides (H-WG(11)-NH(2)) with significant amounts of proteins and glucose. Among the proteins, defensive proteins such as protease inhibitors, proteases and oxidases were abundant. The neurotoxin β-cyanoalanine was also found in the droplets. Despite the presence of cyanogenic glucosides, which release toxic hydrogen cyanide after hydrolysis by a specific β-glucosidase, the only β-glucosidase identified in the droplets (ZfBGD1) was inactive against cyanogenic glucosides. Accordingly, droplets did not release hydrogen cyanide, unless they were mixed with specific β-glucosidases present in the Zygaena haemolymph. Droplets secreted onto the cuticle hardened and formed sharp crystalline-like precipitates that may act as mandible abrasives to chewing predators. Hardening followed water evaporation and formation of antiparallel β-sheets of the peptide oligomers. Consequently, after mild irritation, Zygaena larvae deter predators by viscous and hardening droplets that contain defence proteins and β-cyanoalanine. After severe injury, droplets may mix with exuding haemolymph to release hydrogen cyanide

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Weather conditions and sexual differences affect the foraging behaviour of the insectivorous Cyprus Wheatear, Oenanthe cypriaca (Aves: Passeriformes: Muscicapidae)

Weather conditions (temperature, cloudiness, wind strength and precipitation) had an infl uence on the foraging behaviour of the Cyprus Wheatear (Oenanthe cypriaca). Under cloudy weather conditions the perch-pounce technique from low perches was used more often while under sunny conditions aerial sallying was used more often. A possible reason is the limited visual range from higher perches during high cloudiness. During higher temperatures and low wind speed aerial sallying from higher perches was preferred presumably because the activity of the ectothermic arthropods is strongly infl uenced by temperature and wind speed. Sexual differences existed in foraging behaviour. Females used hop-and-peck on the ground more often than males, and males preferred higher perches. This can be explained by a lower exposure to predators and hence lower predation risk that is especially important for females during the breeding season. Furthermore, males and females may occupy different niches during pre-breeding and incubation

Geographic variation in coal tit song across continents and reduced species recognition between Central European and Mediterranean populations

Intraspecific variation of passerine song may influence mate choice and consequently provides a potential mechanism for premating reproductive isolation in birds. Notable geographic variation of songs is particularly common in widespread bird species, such as the coal tit Periparus ater (Aves, Passeriformes, Paridae) having a large trans-Palearctic distribution range including allopatric populations. In this study, measurement and analysis of 16 song parameters from different Western Palearctic populations showed a relatively uniform song in all continental European and the island populations of Corsica and Sardinia. Song variation among and within these populations seems so variable that there is a broad geographical overlap of acoustic parameters between populations. However, songs from northwest African and Cypriot populations had lowest minimum frequencies and were thus significantly different from continental European, Corsican and Sardinian populations. To test for intraspecific species recognition and potential acoustic isolation of different Palearctic populations, we carried out field playback experiments on Central European coal tit males with songs from populations of the Eurasian (Germany, Latvia, Russian Far East, Japan) and the Mediterranean region (northwest Africa, Cyprus, Corsica, Sardinia). Eurasian song types elicited similar strong territorial reactions in Central European coal tits as local control songs. In contrast, none of the Mediterranean songs evoked any notable reaction. Thus, there is geographic variation in coal tit song across continents that coincides with reduced species recognition between Central European and Mediterranean populations

A subset of chemosensory genes differs between two populations of a specialized leaf beetle after host plant shift

Due to its fundamental role in shaping host selection behavior, we have analyzed thechemosensory repertoire of Chrysomela lapponica. This specialized leaf beetleevolved distinct populations which shifted from the ancestral host plant, willow (Salixsp. Salicaceae), to birch (Betula rotundifolia, Betulaceae). We identified 114 chemosensory candidate genes in adult C. lapponica: 41 olfactory receptors (ORs), eightgustatory receptors, 17 ionotropic receptors, four sensory neuron membrane proteins, 32 odorant binding proteins (OBPs), and 12 chemosensory proteins (CSP) byRNA-seq. Differential expression analyses in the antennae revealed significant upregulation of one minus-C OBP (ClapOBP27) and one CSP (ClapCSP12) in the willowfeeders. In contrast, one OR (ClapOR17), four minus-C OBPs (ClapOBP02, 07, 13, 20),and one plus-C OBP (ClapOBP32) were significantly upregulated in birch feeders.The differential expression pattern in the legs was more complex. To narrow downputative ligands acting as cues for host discrimination, the relative abundanceand diversity of volatiles of the two host plant species were analyzed. In additionto salicylaldehyde (willow-specific), both plant species differed mainly in theiremission rate of terpenoids such as (E,E)-a-farnesene (high in willow) or4,8-dimethylnona-1,3,7-triene (high in birch). Qualitatively, the volatiles were similarbetween willow and birch leaves constituting an "olfactory bridge" for the beetles.Subsequent structural modeling of the three most differentially expressed OBPs anddocking studies using 22 host volatiles indicated that ligands bind with varying affinity. We suggest that the evolution of particularly minus-C OBPs and ORs in C. lapponica facilitated its host plant shift via chemosensation of the phytochemicals frombirch as novel host plant.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Saliva extracts of <i>Z. filipendulae</i> do not inhibit plant cyanogenesis.

<p>Feigl-Anger paper showing HCN emission over time from leaf macerates of <i>L. corniculatus</i> and <i>L. japonicus</i> (wild-type MG-20) incubated with either: insect saliva of <i>Z. filipendulae</i> larvae, water or heat-inactivated saliva as control (latter only on MG-20). When leaf macerates of both <i>Lotus</i> species are mixed with insect saliva, HCN emission increases at a similar rate as the leaf macerate incubated with water or heat-inactivated saliva.</p